Fastin™ Elastin assay kit

Soluble tropoelastins, lathyrogenic elastins and insoluble cross-linked elastins that have been solubilised using hot oxalic acid treatment.

Absorbance peak for TPPS in the dye dissociation reagent is 513nm. However, absorbance settings of 545nm or 550nm also give usable readings. A blue green filter is usually appropriate but filters either side of this should be tested to obtain the highest linear absorbance readings possible.

Fastin measures elastin in solution. Soluble elastin, such as tropoelastin in culture medium, can be used with minimal preparation. Insoluble, cross-linked elastin in tissues and cell layers must first be converted to soluble α-elastin with oxalic acid, which is supplied with the kit.

• Culture medium: swirl gently, collect, centrifuge at 1,500 x g for 10 minutes and assay the clear supernatant (100 µl as standard). Keep serum below 2.5% where possible, and run uncultured medium as a blank.
• Tissues (e.g. skin, lung, blood vessels, heart): use 10–20 mg per extraction in a heat-resistant screw-cap tube. Add 0.25 M oxalic acid: 75 µl per mg of wet tissue, or 250 µl per mg of freeze-dried tissue. Heat at 100 °C for 60 minutes, cool, then centrifuge at 13,000 x g for 10 minutes and keep the supernatant.
• Repeat extractions: repeat the extraction on the residue. Young tissue usually dissolves in one or two extractions; mature tissue may need three. Assay each extract and add the results together.
• Cell layers: detach the cells with trypsin or a non-enzymatic solution and pellet them at 300 x g. Extract the pellet with 400 µl of 0.25 M oxalic acid at 100 °C for 60 minutes.

For a new tissue type, run three sequential extractions on one sample to find how many you need. Extracts can be frozen at −20 °C.

The Fastin QuickStart guide has a one-page flowchart that takes you to the right section of the manual for your sample. Full protocols are in the Fastin manual. You can download both from the Documentation links at the top of this page.

Some tissues may need more than one oxalic acid extraction. Each extraction runs for 60 minutes at 100 °C. Any elastin left in the residue is recovered by adding fresh 0.25 M oxalic acid, in the same volume as before, and repeating. Remember to keep each earlier extract for analysis.

• How many extractions: young or foetal tissue usually dissolves in one or two extractions; mature or aged tissue may need three.
• A new tissue type: run three sequential extractions on a representative sample and assay each extract separately to find out how many you need. Add the results together for total elastin.

If a lot of elastin remains after the third extraction, increase the surface area before extracting. Lung tissue is a common example. Grind or homogenise the tissue, ideally after freeze-drying, or snap-freeze it and grind it while still frozen.

The Fastin dye/elastin labelling interaction is most efficient at neutral pH; for this reason the Fastin Dye Reagent is supplied in a buffered form. The elastin precipitating reagent has a low pH and, if any residual fluid is left, will significantly alter the pH of the sample/dye mixture. This causes a change in the dye colour (from red to green), and a reduction in elastin labelling.

To prevent this occurring, please ensure you remove as much fluid as possible after the precipitation spin, before you add the Fastin Dye Reagent. Drain each tube by inverting it and tapping it onto paper towel, and don't use a pipette (it can dislodge precipitated elastin).

First, check that the standards were prepared correctly. Each tube holds 100 µl in total, made from the 1 mg/ml α-elastin standard and 0.25 M oxalic acid:

• 0 µg/ml (blank): 100 µl oxalic acid
• 50 µg/ml: 5 µl standard + 95 µl oxalic acid
• 100 µg/ml: 10 µl standard + 90 µl oxalic acid
• 250 µg/ml: 25 µl standard + 75 µl oxalic acid
• 500 µg/ml: 50 µl standard + 50 µl oxalic acid

The 0.25 M oxalic acid is made by diluting the 1.0 M stock: 1 part stock to 3 parts water.

Next, check the precipitation step:

• The Precipitating Reagent should be pre-chilled to 4 °C.
• Tubes should stand at 4 °C for 15 minutes before the spin.
• As much reagent as possible should be drained afterwards. Leftover acidic reagent lowers the pH and reduces dye binding (see the question on green staining).

A good standard curve is linear, with R² of 0.99 or better.

A high zero (reagent blank) reading usually means unbound dye was left in the tube. In a typical assay, the zero standard reads between 0 and 0.1.

• After the precipitation spin: drain the tube by inverting it, then tap it firmly onto a single sheet of paper towel to remove the last of the liquid. Don't use a pipette, as it can remove the precipitated elastin.
• After the dye spin: stand the tubes upside down on clean paper towel for 20 minutes to drain. Then wipe any dye droplets from around the tube opening with a cotton bud, without touching the reddish-brown elastin–dye deposit.
• Plate blank: subtract the plate blank (150 µl of Dye Dissociation Reagent in an empty well) from all readings.
• Serum-containing medium: some extra background is expected. Keep serum below 2.5% where possible, and subtract a blank of uncultured medium.

Unopened, all the reagents are stable for 1 year at room temperature. After opening, follow the storage conditions indicated on the label of each component. Do not freeze any kit components.

Lathyrogens are a class of compounds that can cause lathyrism, a condition characterized by connective tissue deformities. These compounds are typically found in food sources, such as legumes of the genus ‘Lathyrus’ which are nutritionally poor, but are sometimes relied upon during times of drought or famine.

These legumes contain the lathyrogenic compound beta-aminopropionitrile (BAPN) (or other aminonitriles) which inhibit the action of the enzyme lysyl oxidase. This disrupts the normal formation of (lysine-derived) cross-links in collagens and elastin – leading to the changes in connective tissue.

The Fastin assay kit can be used to quantify these elastins despite any lathyrogen-induced changes to the normal elastin cross-linking process.